The field of the invention is regulation of cell adhesion and migration.
Plasminogen activators convert the inactive zymogen plasminogen into the broad-spectrum proteolytic enzyme, plasmin (Higgins et al., 1990, Annu. Rev. Pharmacol. Toxicol. 30:91-121; Holden, 1990, Radiology 174:993-1001; Mayer, 1990, Clin. Biochem. 23:197-211). One type of plasminogen activator, designated urokinase-type plasminogen activator (uPA), is a component of the circulatory system and other fluid compartments of the mammalian body.
uPA is the principal cell-associated plasminogen activator and has been implicated in several biological processes including angiogenesis, organogenesis, ovulation, inflammation, cancer, tumor cell invasion and metastasis, atherosclerosis, and other biological and pathological processes characterized by cell migration through physiological barriers such as fibrin and basement membranes (Gyetko et al., 1994, J. Clin. Invest. 93:1380-1387; Gyetko et al., 1996, J. Clin. Invest. 97:1818-1826; Shapiro et al., 1997, Am. J. Pathol. 150:359-369; Dado et al., 1994, Fibrinolysis 8(Suppl. 1):189-203).
uPA is synthesized as a single chain zymogen, designated single chain uPA (scuPA), which exhibits little urokinase activity (Ellis et al., 1987, J. Biol. Chem. 262:14998-15003; Petersen et al., 1988, J. Biol. Chem. 263:11189-11195; Husain, 1991, Biochemistry 30:5707-5805; Colleen et al., 1986, J. Biol. Chem. 261:1259-1266). Activation of scuPA occurs by enzymatic cleavage of scuPA, yielding two-chain uPA (tcuPA). Physiological formation of tcuPA from scuPA is catalyzed primarily by plasmin (Robbins et al., 1967, J. Biol. Chem. 242:2333-2342). scuPA may also be activated by binding of scuPA to the cell-surface receptor, uPAR. In the case of scuPA binding to uPAR, scuPA remains a single chain molecule, but is active (Higazi et al., 1995, J. Biol. Chem. 270:17375-17380).
The activity of uPA is regulated, in part, by plasminogen activator inhibitor-1 (PAI-1), which is a member of the serine protease inhibitor (SERPIN) family of proteins (Kruithof, 1988, Enzyme 40:113-121; Potempa et al., 1994, J. Biol. Chem. 269:15957-15960; Lijnen et al., 1994, Eur. J. Biochem. 224:567-574). PAI-1 is thought to be the most relevant inhibitor of uPA activity in the fluid phase, due to its high second order rate constant of inhibition, 1.7xc3x9710xe2x88x928 Mxe2x88x921.sxe2x88x921, which is higher than any other protease inhibitor(Hekman et al., 1988, Arch. Biochem. Biophys. 262:199-210).
A soluble recombinant form of uPAR, designated suPAR, is known and differs from uPAR by lacking the portion of uPAR that links the receptor to the cell surface. suPAR possesses the same properties as uPAR with respect to binding and activating scuPA and promoting the adhesivity of the uPA-uPAR complex (Higazi et al., 1995, J. Biol. Chem. 270:17375-17380; Higazi et al., 1996, Blood 87:3545-3549).
Binding of scuPA to uPAR enhances urokinase activity of scuPA (Higazi et al., 1995, J. Biol. Chem. 270:17375-17380). Formation of the scuPA-uPAR complex also dampens the capacity of PAI-1 to inhibit scuPA activity, relative to the capacity of PAI-1 to inhibit tcuPA activity (Higazi et al., 1996, Blood 87:3545-3549). Formation of a complex between scuPA and uPAR also alters the regulation of scuPA enzymatic activity by peptide substrates of plasmin and promotes binding of scuPA to vitronectin (Higazi et al., 1996, Thromb. Res. 84:243-252; Higazi et al., 1996, Blood 88:542-551; Wei et al., 1994, J. Biol. Chem. 269:32380-32388; Wei et al., 1996, Science 273:1551-1555; Deng et al., 1996, J. Cell Biol. 134:1563-1571; Stefansson et al., 1996, Nature 383:441-443; O""Reilly et al., 1996, Nature Med. 2:689-692).
A region of uPA, comprising the protein sequence RHRGGS (SEQ ID NO:1) at amino acid positions 179-184, is required for inhibition of uPA activity by PAI-1 (Madison et al., 1990, J. Biol. Chem. 265:21423-21426). Conservation of this sequence among mammalian uPA proteins has been demonstrated (Adams et al., 1981, J. Biol. Chem. 266:8476-8482). Working with a different plasminogen activator protein, namely tissue-type plasminogen activator (tPA), Madison et al. have identified a region of PAI-1 which is involved in inhibition of tPA by PAI-1 (1990, J. Biol. Chem. 265:21423-21426). This region of PAI-1 comprises the sequence RMAPEEIIMDR (SEQ ID NO:2) at amino acids 346-356. It has been postulated that electrostatic interactions between this region of PAI-1 and tPA play a role in stabilizing a tPA-PAI-1 complex. Similarly, it has been postulated that electrostatic interactions between a region of PAI-1 and uPA may contribute to formation of a PAI-1-uPA complex. It has been observed, however, that the scuPA-uPAR complex is less susceptible to inhibition by PAI-1 (Higazi et al., 1996, Blood 87:3545-3549) than is tcuPA or uPAR-bound tcuPA (Higazi et al., 1996, Blood 87:3545-3549; Ellis et al., 1990, J. Biol. Chem. 265:9904-9908).
In addition to inhibiting urokinase activity of uPA, PAI-1 also promotes the internalization and lysosomal degradation of uPA, which involves the xcex12-macroglobulin receptor/low density lipoprotein-related receptor protein (xcex12MR/LRP; Nykjaer et al., 1994, J. Biol. Chem. 269:25668-25676). The complex formed between PAI-1 and tcuPA binds to xcex12MR/LRP with considerably higher affinity than does either component alone. Although it has been demonstrated that the increased affinity of the complex results from an independent contribution of epitopes present in each ligand, a possible conformation-altering effect of PAI-1 upon uPA has not been excluded (Nykjaer et al., 1994, J. Biol. Chem. 269:25668-25676).
When scuPA is bound to uPAR, scuPA is protected from inactivation by PAI-1. Furthermore, binding of scuPA to uPAR inhibits binding of scuPA to xcex12MR/LRP and internalization of scuPA caused by such binding (Nykjaer et al., 1994, J. Biol. Chem. 269:25668-25676; Higazi et al., 1996, Blood 87:3545-3549). Two mechanisms have been postulated for the reduced affinity of uPAR-bound scuPA for xcex12MR/LRP. Nykjaer et al. (supra) proposed that the site at which scuPA contacts xcex12MR/LRP is shielded by uPAR. An alternative mechanism is that binding of scuPA to uPAR induces a conformational change that both promotes scuPA binding to integrin ligands and leads to a loss of the scuPA epitope recognized by xcex12MR/LRP (Higazi et al., 1996, Blood 88:542-551). The latter proposed mechanism is consistent with the observation that soluble scuPA has a higher affinity for xcex12MR/LRP than does tcuPA and with the observation that tcuPA loses affinity for xcex12MR/LRP when the active site of tcuPA is occupied by diisofluoryl phosphate (Nykjaer et al., 1994, J. Biol. Chem. 269:25668-25676).
scuPA bound to uPAR is active, protected from inactivation by PAI-1, and protected from clearance from the cell surface mediated by binding of scuPA to xcex12MR/LRP and subsequent degradation. Furthermore, scuPA that dissociates from uPAR reverts to an inactive conformation and becomes essentially insusceptible to inactivation by PAI-1. Thus, unbound scuPA retains the capacity to rebind to uPAR and revert once again to its active conformation.
There are abundant epidemiological data which indicate that the expression or uPA and uPAR in human tissue correlates with the conversion of cells from a benign to a neoplastic state. Furthermore, expression of uPA and uPAR is associated with a wide variety of common malignancies, and is predictive of future development of those malignancies. Interference with uPA activity by binding an antibody to uPA, by expression of an antisense oligonucleotide complementary to mRNA encoding uPA, or by overexpression of catalytically inactive forms of uPA impede tumor progression in several experimental murine models of human cancers (Ossowski, 1988, J. Cell Biol. 107:2437-2445; Ossowski et al., 1991, Canc. Res. 51:275-281; Kook et al., 1994, EMBO J. 13:3983-3991; Crowley et al., 1993, Proc. Natl. Acad. Sci. USA 90:5021-5025; Jankun et al., 1997, Canc. Res. 57:559-563).
The capacity to regulate uPA activity would enable the practitioner to regulate a number of important human diseases and symptoms thereof. There remains a significant unmet need for compositions useful for modulating the activity of uPA in a mammal, particularly in a human, and for methods of using those compositions to treat pathological conditions attributable to undesirable uPA activity in the mammal. Particularly needed are compositions and methods for promoting internalization and degradation of scuPA which act independently of activation of scuPA by plasmin, independently of binding of scuPA to uPAR, and independently of inactivation of soluble or uPAR-bound scuPA by PAI-1.
The invention includes a composition comprising a peptide having the amino acid sequence X1X2X3X4X5X6X7X8, wherein:
X1 is hydrogen, an amino-terminal blocking group, or one to twenty amino acid. residues;
X2 is an amino acid selected from the group consisting of D, E, H, K, and R;
X3 is an amino acid selected from the group consisting of E and D;
X4 is an amino acid selected from the group consisting of I, L, and V;
X5 is an amino acid selected from the group consisting of I, L, and V;
X6 is an amino acid selected from the group consisting of M;
X7 is an amino acid selected from the group consisting of D, E, H, K, and R; and
X8 is hydrogen, a carboxyl-terminal blocking group, or one to twenty amino acid residues.
In one aspect,
X1 is hydrogen or an amino-terminal blocking group;
X2 is an amino acid selected from the group consisting of D, E, and R;
X3 is an amino acid selected from the group consisting of D and E;
X4 is I;
X5 is I;
X6 is M;
X7 is an amino acid selected from the group consisting of D and E; and
X8 is hydrogen or a carboxyl-terminal blocking group.
In a preferred embodiment,
X1 is hydrogen;
X2 is E;
X3 is E;
X4 is I;
X5 is I;
X6 is M;
X7is D; and
X8 is hydrogen.
In another aspect, the composition of the invention further comprising a pharmaceutically acceptable carrier.
Also included in the invention is a method of affecting a biological process characterized by abnormal cell migration through a physiological barrier. The method comprises administering the composition of the invention to a mammal experiencing the biological process in an amount to affect the biological process.
In one aspect, the biological process is selected from the group consisting of angiogenesis, organogenesis, ovulation, inflammation, cancer, tumor cell invasion and metastasis, and atherosclerosis.
In a preferred embodiment, the mammal is a human.
The invention further includes a method of inhibiting PAI-1-dependent adhesion of a cell to a tissue of a mammal, the method comprising administering to the tissue the composition of the invention in an amount to inhibit adhesion of the cell to the tissue.
In one aspect, the tissue is in vivo in the mammal.
In a preferred embodiment, the mammal is a human.
Also included in the invention is a method of promoting clearance of scuPA from the surface of a mammalian cell, the method comprising administering the composition of claim 1 to the cell in an amount to promote clearance of the scuPA from the cell.
In one aspect, the cell is a human cell.
In a preferred embodiment, the composition is administered in vivo in the human.
Additionally, the invention includes a method of impeding pathological migration of a cell in a mammal. The method comprises administering to the mammal the composition of the invention in an amount effective to impede pathological migration of the cell.
In one aspect, the composition is administered to the mammal at the site of a tumor in the mammal.
In a preferred embodiment, the mammal is a human.
The invention yet further includes a method of inhibiting PAI-1 activity in a tissue of a mammal. The method comprises administering to the tissue the composition of the invention in an amount effective to inhibit PAI-1 activity in the tissue.
In a preferred embodiment, the mammal is a human.
In another preferred embodiment, the composition is administered in vivo in the human.
The invention also includes a kit comprising a peptide having the amino acid sequence X1X2X3X4X5X6X7X8, wherein:
X1 is hydrogen, an amino-terminal blocking group, or one to twenty amino acid residues;
X2 is an amino acid selected from the group consisting of D, E, H, K, and R;
X3 is an amino acid selected from the group consisting of E and D;
X4 is an amino acid selected from the group consisting of I, L, and V;
X5 is an amino acid selected from the group consisting of I, L, and V;
X6 is an amino acid selected from the group consisting of M;
X7 is an amino acid selected from the group consisting of D, E, H, K, and R; and
X8 is hydrogen, a carboxyl-terminal blocking group, or one to twenty amino acid residues, and an instructional material for using the kit.
Also included is a composition comprising a combination of a peptide having the amino acid sequence X1X2X3X4X5X6X7X8, wherein:
X1 is hydrogen, an amino-terminal blocking group, or one to twenty amino acid residues;
X2 is an amino acid selected from the group consisting of D, E, H, K, and R;
X3 is an amino acid selected from the group consisting of E and D;
X4 is an amino acid selected from the group consisting of I, L, and V;
X5 is an amino acid selected from the group consisting of I, L, and V;
X6 is an amino acid selected from the group consisting of M;
X7 is an amino acid selected from the group consisting of D, E, H, K, and R; and
X8 is hydrogen, a carboxyl-terminal blocking group, or one to twenty amino acid residues, and
a thrombolytic agent.
In one aspect, the thrombolytic agent is selected from the group consisting of tissue plasminogen activator, streptokinase, urokinase, the streptokinase derivative and staphylokinase.
Further included in the invention is a composition comprising a combination of a peptide having the amino acid sequence X1X2X3X4X5X6X7X8, wherein:
X1 is hydrogen, an amino-terminal blocking group, or one to twenty amino acid residues;
X2 is an amino acid selected from the group consisting of D, E, H, K, and R;
X3 is an amino acid selected from the group consisting of E and D;
X4 is an amino acid selected from the group consisting of E, L, and V;
X5 is an amino acid selected from the group consisting of I, L, and V;
X6 is an amino acid selected from the group consisting of M;
X7 is an amino acid selected from the group consisting of D, E, H, K, and R; and
X8 is hydrogen, a carboxyl-terminal blocking group, or one to twenty amino acid residues, and
an anti-coagulating agent.
In one aspect, the anti-coagulating agent is selected from the group consisting of an agent which inhibits platelet function, and agent which inhibits the activity of thrombin, and agent which promotes the activity of activated protein kinase C, an anti-thrombin III agent, and a tissue factor pathway inhibitor.